The intracellular signaling mechanisms involved in regulating cardiac myocyte hypertrophy have remained elusive. Studies of proliferating cell types have identified a family of 70 and 85 kDa protein kinases (p70S6K) that phosphorylate ribosomal S6 protein and regulate translation of mRNA, thereby allowing cellular growth and cell cycle progression. Our purpose was to determine whether p70S6K plays a role in 1-adrenergic receptor (beta1-AR) mediated cardiomyocyte hypertrophy. Hypertrophy of neonatal rat cardiac myocytes was induced in culture with 20 muM phenylephrine (PE) and was assessed by 14C-phenylalanine (14C-Phe) incorporation into TCA-precipitable protein and planimetric evaluation of two-dimensional cell area. Immune-complexed p70S6K phosphotransferase activity was measured using synthetic RRRLSSLRA as a substrate. Treatment of myocyte cultures for either 20 min or 24 hours with PE increased p70S6K activity >3-fold. Exposure of cells to PE for 72 hours resulted in a approximately 2-fold increase in 14C-Phe incorporation and a approximately 50% increase in myocyte cell area. Rapamycin, a specific inhibitor of p70S6K activation, completely inhibited the PE-stimulated increases in p70S6K activity, incorporation of 14C-Phe into cardiomyocyte protein, and myocyte cell area. Inhibition by rapamycin was dose dependent, and was reversed by FK506, a related immunosupressant that competes with rapamycin for an FKBP binding site. Wortmannin (1muM) inhibited the PE-induced increase in p70S6K activity, suggesting involvement of a phosphoinositide 3-kinase. It is concluded that beta1-AR stimulation increases p70S6K activity and that activation of p70S6K is required for beta1-AR-mediated hypertrophy of cultured cardiac myocytes.